Process for purifying hepatitis A virus (HAV), virus thus purified and vaccine compositions containing it

ABSTRACT

A process is described for the purification or the hepatitis A virus, which allows one to obtain with good yields a pure product, in which organic material collected by centrifugation after lysis or the culture cells is submitted to gel filtration and successively to ion exchange chromatography.

This is a continuation of application Ser. No. 08/276,780, filed on Jul.18, 1994, U.S. Pat. No. 5,609,851 which is a continuation of Ser. No.08/126,105, now abandoned filed on Sep. 22, 1993, which is acontinuation of Ser. No. 07/894,928, filed Jun. 8,1992 now abandoned.

INVENTION FIELD

A process is described for the purification of the hepatitis A virus(HAV) in which the material from the culture cells, after cell lysis andcentrifugation, is submitted to gel filtration and the thus obtainedeluate is submitted to ion exchange chromatography.

STATE OF THE ART

The hepatitis A virus (HAV) is a icosahedral morphology Picornaviruswith 32 capsomeres on its surface, which presents four importantstructural polypeptides: VP1 with a molecular weight MSW 30.000-33.000,VP2 24.000-27.000, VP3 21.000-23.000, VP4 7.000-14.000.

Said four proteins are the ones responsible for the antigenic viruspower and are therefore the ones which the purification processes tendto put in evidence and to isolate in order to obtain, with a good degreeof purity, an inactivated vaccine. The purification processes seek toeliminate cellular contaminants and growth factors which are employed inthe virus production process.

Various methods for the partial virus purification, both for vaccinationand for virus characterization purposes, have been described. Forexample, through the virus sedimentation by means of a 20% sucrose padand successive centrifugation in a cesium chloride gradient P. J.Provost et al. J. of Medical Virology 19, p. 23-31 (1986)!, throughammonium sulphate precipitation and sedimentation with a 20% sucrose padand cesium chloride gradient Flehmig B. et al., J. of Medical Virology22, p. 7-16 (1987)!, with a lysis buffer, freezing, defrosting,sonication to set the virus free, ultrafiltration with tangential flowand purification in cesium chloride gradient Flehmig et al., The Lancet,May 13, p. 1039 (1989)!, through various clarification cycles andsuccessive freon or chloroform extraction followed by gel filtration,ion exchange chromatography and purification in cesium chloride gradientS. A. Locarnini, Intervirology 10, 300-308 (1978)!.

All the above mentioned processes employ, at least in one step,ultracentrifugation systems and high cost materials such as cesiumchloride, and therefore, although yielding excellent results on a smallscale, are hardly suitable for an industrial production, in whichprocess duration, costs and availability of suitable personnel have tobe taken into consideration. In the European Patent ApplicationEP-A-302692 a process for the purification of hepatitis A virus isdescribed, which employs sonication for the cell lysis, followed byextraction with organic solvents and successive concentration,chromatography on anion exchange resins and, finally, gel filtrationchromatography. Also this process, particularly in view of the use ofsonication and of organic phase extractions and concentrations,presents, on an industrial scale, certain operative difficulties.

DETAILED DESCRIPTION OF THE INVENTION

The process according to the present invention allows to obviate thementioned drawbacks and is therefore a valid contribution to thepurification on an industrial scale and the production of a purified HAVvirus suitable for the use as a vaccine.

Diploid human cells MRC-5 designated by the World Health Organization assuitable for the production of vaccines for human use, cultivated andcollected according to conventional techniques, were used.

The cells were infected with HAV at the 30th passage. After 21 daysincubation, the cell substrate was washed with PBS-A to eliminate asmuch as possible the fetal bovine serum present in the culture mediumand indispensable for the substrate. The infected cells were taken upwith trypsin EDTA following traditional methods and re-suspended inhypotonic buffer (Tris 10 mM, NaCl 10 mM, pH 7.5), this causing celllysis and therefore setting the virus free, and frozen.

At the time of purification the material is defrosted and treated with2% Triton-X-100 for 30 min. at room temperature, stirring about every 5min.; the material is then collected by centrifugation, so as to removethe cell fragments; this treatment allows the solubilization of membranelipids with which the virus is strictly associated. The next step is gelfiltration, employing gel filtration beds of both agarose and dextran,e.g. SEPHAROSE CL-4B resin (Pharmacia) equilibrated in TNE buffer (Tris10 mM, NaCl 150 mM, EDTA 1 mM, pH 7.2-7.6), containing 0.1-0.2%Triton-X-100 or glycine buffer 0.1M with 0.2% deoxycholate, pH 8.5. Theeluated material is collected in 20 ml fractions which are tested forthe presence of HAV by an ELISA assay. With this passage, yields of85-95% are obtained with an approximate eight-fold purity increase(30-50 μg virus per mg of protein).

The eluate obtained in the preceding step is then submitted to ionexchange chromatography employing anion exchange resins, such as e.g.DEAE SEPHAROSE CL-6B resin equilibrated in TNE containing 0.1%-0.2%Triton-X-100; in these conditions the virus is adsorbed on the bed whilepart of the contaminants are not retained.

After washing the column with TNE, to eliminate the detergent, eluitionis performed decreasing the pH and increasing the ion strength. To thisend a phosphate buffer may be employed with a continuous pH gradientfrom 7.4 to 4 and ionic strength from 0 to 0.3M NaCl. The yield in thissecond step is of the order of 50% with respect to the preceding stepand the purity of the collected virus is increased 6 to 10 times (withan average virus contents of 70% on the total protein). The thuspurified material is filtered on a membrane of 0.22 μm porosity andinactivated with 1:2000 formalin at 35° C. for 5 days under continuousstirring.

During the inactivation period, disaggregating treatments are performed,the 2nd day the material is sonicated at 50-60 W 1 sec/ml. The third daythe material is filtered on a 0.22 μm membrane and L-lysine.HCl 25 mM isadded. After inactivation, the suspension is dialyzed against PBS A(1:100 v/v) for about 36 hrs, with an intermediate buffer substitution.After dialysis, the material is submitted to a sterilizing filtrationand the product undergoes all the required controls: sterility,pyrogenicity, inactivation, antigenicity, pH, stability, residualformalin.

EXPERIMENTAL SECTION

MRC-5 cells at the 30th passage in rotating 850 cm² bottles are infectedwith HAV (strain LSH/S ATCC VR 2266) at a 0.5 MOI. After a 20 dayincubation period, the cellular substrate is washed 3 times withserum-free medium maintaining the last washing overnight. The followingday the cells are removed with tripsine-EDTA following traditionalmethods, and suspended again in hypotonic buffer (Tris 10 mM, NaCl 10mM, pH 7.5) 1 ml for each 100 cm² cell culture and frozen.

60 ml of the frozen suspension, deriving from approximately 5.700 cm²culture are defrosted and treated with a non ionic detergent (2%Triton-X-100) for 20 to 30 minutes at room temperature under moderatestirring every 5-10 minutes. The sample is centrifuged at 400 g for 10minutes while cooling to remove cellular fragments. The supernatant ispurified through gel filtration on a agarose resin (SEPHAROSE CL4B resinPharmacia)column 5×90 cm (K 50/100 column, Pharmacia) equilibrated withTris 10 mM, NaCl 150 mM, EDTA 1 mM buffer, pH 7.4, containing 0.1%Triton-X-100 at a 75 ml/h flow rate. The eluted material is collected in20 ml fractions which are tested for the presence of HAV by a ELISAassay. The HAV containing fractions are collected, obtainingapproximately 400 ml. This material is further purified by ion exchangechromatography seeding about 200 ml, at a flow rate of 100 ml/h on aanionic resin (SEPHAROSE CL6B resin Pharmacia) column 5×5 (column XK50/30 Pharmacia) which had previously been equilibrated in Tris 10 mM,NaCl 150 mM, EDTA 1 mM, pH 7.4 buffer containing 0.1% Triton-X-100.Under such conditions the virus is adsorbed on the matrix. The matrix iswashed with Triton-X-100 free buffer to remove the detergent and thevirus is eluted at a flow of approximately 160 ml/h, applying acontinuous pH gradient and ionic strength, starting from pH 7.4 and NaCl0 mM to pH 4 and NaCl 10.3M. The eluted material is collected infractions of about 10 ml and the fractions found positive for thepresence of HAV at a ELISA assay are put together.

A virus content of 70% on the total protein is thus obtained. The thuspurified material is filtered on 0.22 μm porous membrane and inactivatedwith formalin 1.2000 at 35° C. for 5 days under continuous stirring.During the inactivation period, disaggregation treatments are performed:on the 2nd day the material is sonicated at 50-60 W per 1 second/1 ml;on the 3rd day it is filtered on a 0.22 μm membrane and L-lysine.HCl 25mM is added. After inactivation, the suspension is dialyzed againstPBS-A (KCl 2.7 mM, KH₂ PO₄ 1.5 mM, NaCl 137 mM, NaH₂ PO₄ 8.1 mM, pH 7.4)in a 1:100 v/v ratio for 36 hours with an intermediate buffersubstitution. After dialysis, the material undergoes a sterilizingfiltration and is then submitted to the usual controls for sterility,pyrogenicity, inactivation, antigenicity, pH, stability and residualformalin.

Hepatitis A virus, strain LSH/H, purified as outlined above wasdeposited with the American Type Culture Collection (ATCC),12301Parklawn Dr., Rockville, Md., 20852, on Oct. 24, 1989. The deposit wasmade pursuant to the Budapest Treaty. The deposit was assiged ATCCaccession number VR2266.

What is claimed is:
 1. Hepatitis A virus (HAV) strain LSH/S deposited asATCC VR
 2226. 2. HAV virus purified and inactivated, obtained accordingto the process as follows:a) lysing cells infected with HAV strain LSH/Sin the presence of a first concentration of detergent; b) removing cellfragments to produce a supernatant; c) performing gel filtration of thesupernatant in the presence of a second concentration of detergent; d)submitting the eluate obtained in step c) to ion exchangechromatography; and e) eluting the virus adsorbed in step d) using adetergent-free elution buffer.
 3. A vaccine composition containing thevirus according to claim 2.